Martin et al. (2005, Nat. Med., publ. online Jan. 30, 2005, doi 10.1038i)
Analysis of sialylated glycans from embryonal stem cells was produced in USA and reported for human embryonal stem cell lines, such cell lines have been reported to be contaminated with N-glycolylneuraminic acid (NeuGc) and amounts of both NeuGc and N-acetylneuraminic acid (NeuAc) have been quantitated. The scientists further discussed cell culture materials containing NeuGc and causing contamination of the cultivated cell lines and for handling the problem by using alternative recombinant protein materials and especially heat inactivated human serum not originally containing NeuGc.
The inventors of the present invention were able to find new sources of potential NeuGc and other sialyl-glycan contaminations. The present invention further describes specific sialyl-glycan structures from early human cells. The inventors were also able to describe specific protein reagents involved in cell production processes, which need to be controlled with regard to glycosylation, especially albumin, gelatine, and antibody reagents.
Varki, U.S. patent document 2005
The patent document describes monosaccharide NeuGc analysis from foods and other materials. There are specific claims for the proportion of NeuGc of the sum of NeuGc and NeuAc, especially in food materials. The document further describes anti-NeuGc antibodies present in patients and production of antibodies involving oxidation of the glycerol tail of NeuGc. The incorporation of NeuGc to a cultured endothelial (cancer) cell line was studied in serum containing culture by adding free NeuGc.
Analysis of CD34+ Hematopoietic Stem Cell Materials with Regard to NeuAcα3Galβ4
NeuAcα3Galβ4 structures have been previously indicated to be present in human cord blood CD34+ hematopoietic cells by the use of a specific monoclonal antibody (Magnani, J., et al., U.S. Pat. No. 5,965,457.). The invention claims all CD34+ cells and especially ones from cord blood and bone marrow
The inventors of the present invention were able to analyse, in human stem cell and cord blood cell populations, the presence of both NeuAcα3 and NeuAcα6 structures and even NeuGcα3/6, and larger sialylated structures, including also information about the glycan core structures by which the glycans are linked to the cell. The present invention is in a preferred embodiment directed to analysis of at least two or several sialylated terminal epitopes or at least one whole glycan structure. In a preferred embodiment the sialic acid analysis of cord blood cells is directed to multipotent cell populations, which are not CD34+ hematopoietic progenitor cells. Preferably the analysis includes analysis of the core structures of N-linked glycans since Magnani et al. (U.S. pat.) do not describe the core structures by which the glycans are linked to the cells.
Desialylation and Resialylation of Cells According to the Invention
Change of sialylation by desialylation and resialylation with specific sialyltransferases has been reported for red cells in order to analyze binding specificities of influenza virus (Paulson, J., et al.).
Partial desialylation and alpha-6-resialylation with CMP-NeuAc-fluorescein of human peripheral blood and bone marrow aspirate-derived CD34+ cells has been reported, the peripheral blood cells having been released by GM-CSF and most of the subjects being under cancer therapy (Schwarz-Albiez, Reihard et al., 2004, Glycoconj. J. 21 451-459). The large variations in results may be due to therapy and GM-CSF. The method used does not reveal real quantitation of sialic acid types due to limited specificity of especially the sialyltransferase used, nor are the possible carrier structures of the sialic acids revealed. The modifications of sialic acid would likely further affect the acceptor specificity of the sialyltransferase used and thus the structures labelled. The present invention is especially directed to α3-sialylation of the specific carrier structures.
Removal of NeuGc from pig xenotransplant tissue and resialylation by NeuAc and sialyltransferase has been also suggested (WO02088351)). That work was not directed to stem cells, nor human stem cells directed methods, nor were the methods used specified, although this is essential for applications in these cells. The xenotransplantation idea is not relevant to present invention due to tissue and species specificity of glycosylation. A patent application (WO2003/105908) describes possible sialidase and sialyltransferase reactions for certain NK/lymphocyte cell lines in a patent application also discussing separately stem cells. The results reveal that the possible reactions vary between cell lines of the same type and are not expected/predictable under the conditions used in the work, possibly partially due to nature of the cells and specificities of enzymes, further the reaction conditions of sialyltransferase without CMP-sialic acid are not described by the invention.
Methods of removal of terminal Gal or GalNAc from human red cells have also been described as well as galactosylation of human platelets in the context of cryopreservation induced changes in human platelets (Zymequest; Science 2004).
None of the reports describe the specific expression of the preferred sialylated N-glycan structures of human stem cells and cord blood cells. It is generally known that glycosylation is cell type specific, and this has been further indicated by the present invention. It cannot be known in advance and based on prior art if the cells contain sialic acid residues removable by specific sialidase enzymes or specific acceptor sites for specific sialyltransferases. Specific sialyltransferases according to the invention, especially recombinant human sialyltransferases controlled with regard to glycosylation, are preferred for the process described in the present invention. The present invention is further directed to the synthesis of the specific sialylated glycan structures according to the present invention, which have not been described in the background publications.
NeuGc Recognizing Antibodies
Production and purification of polyclonal antibodies, which recognize NeuGc has been published by Varki and colleagues, Martin et al 2005 (antibody production WO2005010485). The specificity appears to be broad and glycerol side chain of the antigen is removed further restricting the specificity. Several antibodies against NeuGc structures has been published but suitability of these against stem cells is not known.
Removal of NeuGc from Bovine Serum Proteins
Acid Hydrolysis for Desialylation of Aglycoprotein
Acid hydrolysis method has been described for desialylation of a glycoprotein called fetuin (Spiro et al., 1982) using 12.5 mM sulfuric acid at 80 degrees of Celsius. This method was designed to release sialic acid quantitatively from the glycoprotein. However, the amount of degraded protein was not analyzed. The inventors realized that under conditions of Spiro et al. the protein polypeptide chain of fetuin and other proteins are heavily degraded to smaller molecular weight fragments. The present invention revealed novel methods, which allow preserving the protein intact and production specific sialylation levels and structures on glycoproteins.
The method of Spiro et al has not been described for NeuGc and/or α6-linked sialic acids (the invention was revealed to be effective for α6-sialylated proteins). The present invention is especially directed to controlled reduction and/or increase of NeuGc on glycans of a protein and control of α6-sialylated structures on protein. The preferred protein according to the invention includes bovine serum proteins, though it is realized that the methods are suitable for modification of other proteins, too.
A. When polyvalent Neu5Gc conjugate was added to the culture medium, human bone marrow derived MSC took it inside the cells, specifically increasing the staining of the cells by Neu5Gc mAb.
B. The staining pattern with Neu5Gc mAb was particulate in appearance, indicating that the Neu5Gc conjugate was localized in distinct compartments of the cells.